In many applications, it is desirable to detect the interaction between substances in a sample. For instance, the detection of the interaction between protein and ligand, protein and glycoprotein, or proteins is very useful for diagnosis of diseases, studies on biotechnology, and development of agriculture and pharmaceuticals.
Many methods for detecting the interaction between molecules have been developed. In the detection of the interaction of protein, SAR-by-NMR and Affinity-NMR are used. The SAR-by-NMR is a method which detects the interaction between target protein which has been subject to 15N isotope labeling and ligand from the change in chemical shift to advance molecular design of ligand with stronger interaction (for instance, Patent Document 1: WO 97/18469, Non-Patent Document 1: Yoshiki Yamaguchi and Kazuo Shimada, “A new approach to a study on novel drug discovery using nuclear magnetic resonance”, protein, nucleic acid, enzyme, 45 (2000) 895). The Affinity-NMR is a detection method which uses the change in relaxation time of a signal produced from the change in translational motion when ligand of low molecular weight is bound to target protein.
Interaction measurement by nuclear magnetic resonance alone has some drawbacks. First, since the SAR-by-NMR must analyze the structure of labeled protein, there is an upper limit (40 KD or below) of analyzable molecular weight. Since a large amount of isotope labeled protein is necessary, the solubility of target protein is restricted to limit the kinds of measurable proteins. Large numbers of isotope labeled samples are very expensive.
Second, since the Affinity-NMR does not require a large amount of isotope labeled protein, measurement is relatively inexpensive. It can detect only the binding strength between protein and ligand. The position of an active region or a binding region and the structure of target protein cannot be found. It is difficult to obtain structural information as a guide for developing more excellent ligand.
A protein-protein (glycoprotein) complex and huge protein are measured by combining selective isotope labeling performing protein synthesis using a medium in which specified amino acid has been subject to isotope labeling with nuclear magnetic resonance (see Non-Patent Document 2: J. Wigelt, M. Wikstrom, J. Schultz, M. J. P. van Dongen, Combinational Chemistry & High Throughput Screening, 5 (2002) 623). Only a resonant signal from the atom on the specified amino acid residue of a sample which has been subject to the selective isotope labeling is observed to make it possible to relegate the residue of protein of high molecular weight.
To perform the selective isotope labeling, a medium in which specified amino acid has been subject to isotope labeling is prepared to perform protein synthesis using sophisticated technique. The difference in experience and skill among sample preparers is largely affected to increase the sample preparing cost.
Most of the prior art methods for detecting the binding between molecules using nuclear magnetic resonance compare the measured results before and after binding target molecules. Measurement of the process of interaction is very difficult.
As experimental means changing the state of electronic spin to detect a resonance signal, ESR is widely used. The ESR uses a wide frequency bandwidth from some hundreds of MHz to some tens of GHz. Energy provided to unpaired electron by the ESR in the frequency bandwidth is significantly smaller than chemical bonding, hydrogen bond, and van der Waals force. Its characteristic is not changed by providing pulses to a sample for a long period of time. In this point, the ESR can be a non-destructive measuring method.
In ultraviolet Ramann spectroscopy which observes the response of a sample by using a high-energy pulse laser, denaturation of sample protein can be brought about when the output of the pulse laser is too strong. Application of a laser having a frequency higher than that of an infrared ray can bring about the structural change of protein. Since the applied energy is near binding energy of atoms structuring protein and rotational energy of an atomic group, the energy cannot be selectively supplied only to the specified portion. In the experimental method of analyzing the response of the entire sample to an incident light of Ramann spectroscopy, selective observation of the specific portion is difficult.
In the function and structural analysis of protein, the need for targeting protein of higher molecular weight and for analyzing a ligand-protein complex is on the increase. A technique for changing and observing the specified portion of protein has not been established.
[Patent Document 1] WO 97/18469
[Non-Patent Document 1] Yoshiki Yamaguchi and Kazuo Shimada, “A new approach to a study on novel drug discovery using nuclear magnetic resonance”, protein, nucleic acid, enzyme, 45 (2000) 895)
[Non-Patent Document 2] J. Wigelt, M. Wikstrom, J. Schultz, M. J. P. van Dongen, Combinational Chemistry & High Throughput Screening, 5 (2002) 623)